Background Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by self-limited attacks of fever with serosal, synovial, or cutaneous inflammation. In 1992 we mapped the FMF locus to chromosome 16p13.3, and in 1997 we isolated the underlying gene, MEFV, and demonstrated that it is expressed predominantly in granulocytes. During the 4 years leading up to the current reporting period, we have (1) performed mutational studies that expanded the spectrum of FMF-associated MEFV mutations, and documented high carrier frequencies in several ethnic groups; (2) demonstrated MEFV expression in myeloid bone marrow precursors and in cytokine-stimulated monocytes, but minimal expression in lymphoid cells; (3) shown in transfected cell lines that the full-length FMF protein (denoted pyrin) associates with microtubules and the actin cytoskeleton; (4) identified 2 pyrin-interacting proteins by the yeast two-hybrid assay: apoptosis-associated specklike protein with a caspase recruitment domain (ASC), and proline, serine, threonine phosphatase interacting protein (PSTPIP1); (5) cloned the mouse and rat orthologs of MEFV, and demonstrated that protein expression in peritoneal monocytes is induced by LPS, IL-4, and IL-10, and is ablated in NF kappa B knockout mice and in mice genetically unresponsive to LPS; and (6) bred mice with a targeted disruption of Mefv that express a truncated mouse pyrin, and demonstrated that macrophages from these mice exhibit a defect in caspase-8-mediated apoptosis. Results of the Last Year Characterization of mice expressing a truncated form of pyrin: Pyrin truncation mice exhibited exaggerated temperature responses to sublethal doses of bacterial endotoxin. LPS challenge also led to increased mortality due to endotoxic shock: death rates were 29%, 25%, and 65% among wild type, heterozygous, and homozygous pyrin-truncation mice, respectively. Peritoneal macrophages from homozygous pyrin mutant mice produced markedly more mature IL-1 beta, relative to heterozygous and wild-type controls, when stimulated with LPS and IL-4, which normally induce pyrin. Moreover, LPS-stimulated soluble IL-1 beta, as measured by ELISA in culture supernatants, was significantly higher for homozygous truncation peritoneal macrophages than for wild-type. Retroviral overexpression of full-length pyrin in a mouse monocytic cell line led to decreased IL-1 beta processing, both by Western blots of cell lysates, and by ELISA of culture supernatants, whereas the truncated pyrin expressed in mutant mice had a hypomorphic effect. Homozygous mutant peritoneal macrophages exhibited not only increased IL-1 beta processing, but augmented caspase-1 activation. The link between pyrin and caspase-1 appears to be ASC, which binds pyrin through homotypic N-terminal PYRIN domain interactions, and which also binds and activates caspase-1. We hypothesize that binding of full-length pyrin to ASC prevents ASC from activating caspase-1, while the truncated form of pyrin does not interfere with this pathway. We also extended our finding of impaired monocyte apoptosis in pyrin truncation mice, demonstrating that resistance to apoptosis is not reversed by neutralizing antibodies against IL-1 beta, and that caspase-9-dependent staurosporine induced cell death is not impaired in these mice. Analysis of arthritis-associated mutations in PSTPIP1, a pyrin-binding protein: We have previously shown that PSTPIP1 binds pyrin in the yeast two-hybrid assay, and have confirmed this by coimmunoprecipitation, and by immunostaining of endogenous protein in peripheral blood monocytes. Moreover, we have shown that the binding of PSTPIP1 to pyrin varies with the phosphorylation status of PSTPIP1. Recently, another group demonstrated that 2 mutations in PSTPIP1 cause the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA). We have subsequently found that, relative to wild-type, these PSTPIP1 mutants (A230T and E250Q) were hyperphosphorylated when coexpressed with c-Abl kinase, and that the mutants showed increased pyrin-binding by immunoprecipitation. In PAPA syndrome, we hypothesize that increased binding may lead to sequestration of pyrin, thus interfering with pyrin?s antiinflammatory functions. Gene expression profiles in monocytic cell lines transfected with MEFV: We have begun gene expression profiling in human monocytic cell lines transfected with a full-length MEFV expression construct. Our initial experiments have utilized the U937 cell line, which shows only low levels of endogenous MEFV expression. RNAs from U937 cells transfected with MEFV or empty vector have been fluorochrome-labeled and hybridized to a cDNA microarray of 8,400 known human genes. Preliminary results to date indicate upregulation of CD46, mitogen-activated protein kinase kinase 7, and apoptosis inhibitor 5. Genes that were significantly down-regulated include ASC, BCL-X, CD27, C/EBP, beta 1 defensin, MIP1 alpha, RANTES, properdin factor D, MHC Class II, the beta subunit of the IL-10 receptor, and several interferon-inducible genes. Mutational and treatment studies in another recessively inherited febrile disorder, the hyperimmunoglobulinemia D syndrome (HIDS): In the course of evaluating patients referred with possible FMF, we have screened for HIDS, which is caused by mutations in mevalonate kinase (MVK). In 2 such cases we have discovered novel disease-associated MVK mutations. One mutation leads to skipping of exon 3, and the second is a missense substitution (S329R). Because both patients had failed other treatments, we undertook an open-label trial of etanercept, the soluble TNFR p75:Fc fusion protein. In both patients etanercept reduced the frequency of febrile attacks and significantly ameliorated clinical symptoms, suggesting a possible role for TNF in the pathogenesis of HIDS attacks. Conclusions and Significance Our findings contribute to a growing body of data demonstrating an important role for pyrin in the regulation of inflammation and apoptosis. Earlier in vitro studies suggested that pyrin may modulate IL-1 activation through ASC and caspase-1, and our data on knockout mice now establish the physiologic relevance of pyrin as a negative regulator of this pathway, and indicate that pyrin?s proapoptotic effect is independent of IL-1. The increased sensitivity of pyrin truncation mice to endotoxin suggests that increased responsiveness to bacterial products may account for the selection of hypomorphic variants in man, and that the episodic nature of FMF may be related to transient bacteremias. Pyrin-related proteins also appear to play an important role in other human inflammatory diseases. PSTPIP1, one of the pyrin-interacting proteins we identified by yeast two-hybrid assays, is mutated in PAPA syndrome, and disease-associated mutations markedly affect the strength of the interaction, suggesting that PSTPIP1 may be an upstream regulator of pyrin. Mutations in CIAS1, which encodes another protein with an N-terminal PYRIN domain, have already been shown to cause two inflammatory disorders (Muckle-Wells syndrome and familial cold autoinflammatory syndrome), and, in the current reporting period, Project 1 Z01 AR041123-04, we have found that mutations in this gene also cause neonatal onset multisystem inflammatory disease (NOMID). During the next year, our objectives will be 1) to continue patient-oriented studies aimed at gene-discovery and elucidating disease mechanisms; 2) to extend studies of animal models, including pyrin truncation mice, a new line of pyrin-null mice, and 3 lines of pyrin knockin mice; and 3) to continue cellular and molecular biologic studies, including analyses of the role of pyrin in NF kappa B signaling and structural studies of pyrin and related proteins.